Method for producing a nematocidal composition by heat treating a pH-adjusted fermentation broth

ABSTRACT

The present invention is directed to a method for producing a new nematocidal composition particularly useful against plant parasitic nematodes and also a process to prevent damage resulting from nematode infestation. The method for production of the composition involves heating a pH-adjusted fermentation broth of microorganisms to a temperature of at least about 100° C. for at least about 15 minutes. Preferably, the microorganism is  Gibberella fujikuroi, Streptomyces erythraeus, Bacillus sphaericus, Bacillus thuringiensis  or  Fusarium moniliforme.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/196,257, filed Apr. 11, 2000.

FIELD OF THE INVENTION

The present invention is directed to a method for producing a newnematocidal composition particularly useful against plant parasiticnematodes and also a process to prevent damage resulting from nematodeinfestation. The method for production of the composition involvesheating a pH-adjusted fermentation broth of microorganisms to atemperature of at least about 100° C. for at least about 15 minutes.

BACKGROUND OF THE INVENTION

Plant parasitic nematodes cause serious economic damage to manyagricultural crops around the world. The nematodes in this group aremicroscopic worms and are, in general, obligate parasites of plants.They feed mostly on the roots of host plants; however, several generaare known to parasitize above-ground parts including stems, leaves andflowers as well. Almost all the plant species of economic importance aresusceptible to infection by some species of nematodes (notableexceptions are in the marigolds and asparagus). For example, root knotnematodes (RKN), (Meloidogyne spp.) are capable of parasitizing morethan 3,000 species of crop plants. These plants include agronomic crops,vegetables, fruits, flowering trees and shrubs. Nematodes reportedlycause crop loss equivalent to more than six billion dollars in theUnited States alone and more than one hundred billion dollars around theworld.

The symptoms due to parasitic nematode injury vary widely depending onthe plant host, the nematode species, age of the plant, geographicallocation and climatic and external environmental conditions. In general,an overall patchy appearance of plants in a field is consideredindicative of nematode infestation. More specifically, nematode injuryresults in galling of the roots (abnormal swelling in the tissue due torapid multiplication of cells in the cortical region) caused by speciesof root knot (Meloidogyne spp.) and cyst (Heterodera spp.) nematodes,lesions (localized, discolored areas) caused by lesion nematodes(Pratylenchus spp.), suppression of cell division resulting in stubbyroots (Trichodorus spp.), growth abnormalities including crinkling ortwisting of above-ground parts (Aphelenchoides spp.), and even cellnecrosis (death) in some cases. Plant parasitic nematodes may beendoparasitic in nature, as in the case of the root-knot and lesionnematodes, or ectoparasitic as in the dagger nematode (Xiphinema spp.)and lance nematode (Hoplolaimus spp.). Nematodes can be vectors of plantviruses and are also known to induce disease complexes predisposingplants to infection by other plant pathogenic fungi and bacteria.

Chemical nematocides, either soil fumigants or non-fumigants, have beenin use for many years and are among the few feasible options forcountering nematodes. At present, the process involves repeatedapplications of synthetic chemicals to the ground prior to planting thecrop. These chemicals are extremely toxic to organisms besides nematodesand many of them may pose serious threats to the environment. With therenewed emphasis on clean water and air by environmental groups andgovernmental agencies, and the detection of many of these activeingredients or the metabolites thereof in ground water and severalnon-target organisms, there has been serious concern as to themanufacture and/or use of these chemicals. One of the most effective,economical, and widely used nematocides, DBCP(1,2-dibromo-3-chloropropane), found in ground water has been judged toinduce male sterility and possible carcinogenesis. Another widely usedchemical, EDB (ethylene dibromide), has also been found in ground water.

Yet another very common insecticide-nematocide, aldicarb(2-methyl-2-(methylthio)-propionaldehyde-O-(methylcarbamoyl)oxime), hasbeen found to be acutely toxic. Aldicarb has been found in ground waterin several regions of United States. Carbofuran(2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) and 1,3-D(1,3-dichlorpropane), two very commonly used nematocides, are underspecial review by EPA because of their avian toxicity and possiblecarcinogenic effects. More recently, the decision by EPA to limit andeventually discontinue the use of the soil fumigant, methyl bromide, foragricultural purposes presents a threat to the efficiency and quality ofagricultural production in the United States.

Natural isolates such as N-acetyl-D-glucosamine, which may be derivedfrom microorganisms which are the waste products of industrialfermentation processes, have been disclosed as nematocidal in U.S. Pat.No. 5,057,141.

Biopesticides have been developed as an alternative to chemicalpesticides. They are obtained by fermentation and can be used either ascrude biomass or purified. Typically, fermentations are carried out attemperatures in the range of 20-40° C. For example, submergedfermentation at 28-30° C. of Paecilomyces fumosoroues fungal isolateATCC No. 20874 produces fungal biomass for control of nematodeinfestation as disclosed in U.S. Pat. No. 5,360,607; whole fermentationbroth from fermentation at 28° C. of Streptomyces thermoarchaensis NCIB12015 is disclosed as nematocidal in U.S. Pat. No. 5,182,207;fermentation broth obtained from fermentation of Streptomycescyaneogriseus noncyanogenus NRRL 15773 at 28° C. is effective againstnematodes as disclosed in U.S. Pat. No. 5,439,934; and fermentationbroth obtained by fermentation of the fungus Myrothecium verrucaria attemperatures of from 25 to 30° C. is disclosed as nematocidal in U.S.Pat. No. 5,051,255.

Heating of an infested biomass, as disclosed in U.S. Pat. No. 4,229,442,at a temperature of at least 125° C. with water in an amount rangingfrom 1-5 times the dry weight of the biomass, may combat nematodes.

However, there is still a need for the development of new and effectivenematocides. It is therefore an object of this invention to provide amethod for the production of nematocidal compositions which areeffective and inexpensive.

BRIEF SUMMARY OF THE INVENTION

The invention is directed to a method of producing a nematocide fromfermentation broths of microorganisms by heating a fermentation broth toa temperature of at least about 100° C. after adjusting the pH to aboutpH 2 or below, or about pH 8 or above. The nematocide thus produced canbe used to prevent plant damage and/or limit the growth of nematodes.

The present invention is directed to a method for improvingbiopesticidal activity of materials produced by fermentation comprisingthe steps of:

fermenting a bacterium or fungus to obtain a fermentation broth;

adjusting the pH of said fermentation broth to a pH of below about 2.5with a biologically acceptable acid, or to a pH of above about 8 with abiologically acceptable alkali or base; and

heat treating said fermentation broth to a temperature of at least about100° C. for at least about 15 minutes, then cooling to ambienttemperature to obtain a pH-adjusted, heat treated composition havingimproved biopesticidal activity;

with the proviso that said fungus is not Myrothecium verrucaria when thepH of said fermentation broth is adjusted to a pH of below about 2.5.

The invention is also directed to a composition comprising

a heat-treated, pH adjusted fermentation broth of a fungus or bacteriumselected from the group consisting of Gibberella fujikuroi, Streptomyceserythraeus, Bacillus sphaericus, Bacillus thuringiensis and Fusariummoniliforme;

wherein said broth is pH adjusted either to a pH of below about 2.5 witha biologically acceptable acid, or to a pH of above about 8 with abiologically acceptable alkali or base;

and wherein said broth is heated to a temperature of at least about 100°C. for at least about 15 minutes, then cooled to ambient temperature.

The invention is also directed to a method for control of nematodes onplants which comprises the step of administering to the locus, soil orseed of plants in need of such treatment, a nematocidally effectiveamount of a composition formed by heating a pH adjusted fermentationbroth from a fungus or bacterium;

wherein said broth is adjusted to a pH of below about 2.5 with abiologically acceptable acid, or to a pH of above about 8 with abiologically acceptable alkali or base;

and wherein said broth is heated to at least 100° C. for at least 15minutes;

with the proviso that said fungus is not Myrothecium verrucaria when thepH of said fermentation broth is adjusted to a pH of below about 2.5.

The composition may be administered at a rate of from about 1 to about200 pounds per acre.

The invention is also directed to a method for producing a nematocidalcomposition comprising the steps of:

a) fermenting a bacterium or fungus to obtain a fermentation broth;

b) suspending said broth in an aqueous solution;

c) adjusting the pH of said broth in aqueous solution to a pH belowabout pH 2.5 with a biologically acceptable acid;

d) heating the pH-treated broth of step c) to a temperature of at leastabout 100° C. for at least about 15 minutes, then cooling to ambienttemperature; and,

e) recovering said composition;

with the proviso that said fungus is not Myrothecium verrucaria.

Preferably, the pH is adjusted below about 2; and most preferably, thepH is adjusted below about 1.5.

The pH of the composition formed in step d) may be adjusted to a rangeof about pH 4 to about pH 8. The fermentation broth of step a) may havewater-soluble biomass and water-insoluble biomass; and the water-solublebiomass may be separated from said water-insoluble biomass, and thewater-insoluble biomass may be suspended in aqueous solution in step b).

The invention is also directed to a method for producing a nematocidalcomposition comprising the steps of:

a) fermenting a bacterium or fungus to obtain a fermentation broth;

b) suspending said broth in an aqueous solution;

c) adjusting the pH of said broth in aqueous solution to a pH aboveabout pH 8 with a biologically acceptable alkali or base;

d) heating the pH-treated broth of step c) to a temperature of at leastabout 100° C. for at least about 15 minutes, then cooling to ambienttemperature;

e) recovering said composition.

Preferably, the pH is adjusted above about 9 and most preferably the pHis adjusted above about 10.

The pH of the composition formed in step d) may be adjusted to a rangeof about pH 4 to about pH 8. The fermentation broth of step a) may havewater-soluble biomass and water-insoluble biomass; and the water-solublebiomass may be separated from said water-insoluble biomass, and thewater-insoluble biomass may be suspended in aqueous solution in step b).

For the practice of any aspect of this invention, at least one compoundsuch as herbicides, antimicrobials, fungicides, insecticides, plantgrowth regulators or nutrients may be added to the heat-treated, pHadjusted compositions.

DETAILED DESCRIPTION OF THE INVENTION

To produce a nematocidal preparation, a substantial amount of biomass isprepared by submerged fermentation of a bacterium or fungalmicroorganism. Preferred examples of microorganisms include Baccillusspp., Myrothecium spp., Gibberella spp., Streptomyces spp., and Fusariumspp. More preferred are the Myrothecium spp. and the Bacillus spp.

More specifically, the composition can be obtained from Gibberellafujikuroi (ATCC 12616 or 14164), Streptomyces erythraeus (also known asSaccharopolyspora erythraea, ATCC 11635 or 31772), Bacillus sphaericus(ATCC 4978), Bacillus thuringiensis such as Bacillus thuringiensisisraelensis (ATCC 35646), Bacillus thuringiensis kurstaki (ATCC 33679)and Bacillus thuringiensis Berliner (ATCC 19268) and Fusariummoniliforme (ATCC 10052). The composition can be obtained from anybacterium or fungal microorganism, since the method breaks down the cellwall of the microorganism. It is believed that the cell walls ofmicroorganisms (branched polysaccharides) are partially or completelyhydrolyzed under the conditions described herein, resulting in theformation of nematocidal hydrolysis products. Therefore, the method isnot microorganism-specific.

The material is suspended or dissolved in an aqueous solution,preferably water, and is hydrolyzed by acidification to a pH below aboutpH 2.0 by means of a biologically acceptable acid such as, for example,sulfuric, hydrochloric or phosphoric acid, or organic acids such asacetic or formic acid, and heated at a temperature of at least about100° C. for a period of at least about fifteen minutes. The materialthat is treated in this manner is preferentially the water-insolubleportion of the biomass from the fermentation, and the insoluble materialmay be separated from the water-soluble portion of the fermentationproduct, but separation is not necessary. The entire fermentation brothproduced in the fermentor may be treated by the process of thisinvention. After the mixture has been acidified and heated, it willoften be appropriate to readjust the pH of the mixture to a more neutralpH to enhance safety of handling and to reduce the risk of damage toplants which would be treated with the preparation. Said pH adjustmentmay be carried out by addition of any biologically-acceptable alkali orbase, such as sodium hydroxide, potassium hydroxide, magnesium oxide,magnesium hydroxide, calcium oxide, calcium hydroxide, or ammoniasolution (ammonium hydroxide).

Alternatively, the treatment can also be achieved by raising the pH toabove about pH 8 by means of alkalization with biologically-acceptablealkali or base such as, for example, sodium hydroxide, potassiumhydroxide, magnesium oxide, magnesium hydroxide, calcium oxide, calciumhydroxide, or ammonia solution (ammonium hydroxide), then heating theresulting mixture as above. Again, after heating, it may be appropriateto adjust the pH of the resulting mixture to a more neutral pH by meansof a biologically acceptable acid such as, for example, sulfuric,hydrochloric or phosphoric acid, or organic acids such as acetic orformic acid.

The heating step may be performed at a pressure above atmosphericpressure, if necessary. For example, elevated pressure may be achievedby heating within an autoclave.

The product thus produced is used to protect plants or control thegrowth of nematodes by applying it in solid form or as a suspension inaqueous solution, preferably water, directly to the surface or the rootzone of the soil in which the plants are grown.

An advantage of the method which we have discovered is that thenematocidal composition is inexpensive and safe. The materials employedin the process include the fermentation broth, comprising thewater-insoluble solids contained in such broth, which may be wastesolids from any industrial fermentation process, such as a fermentationcarried out to prepare pharmaceutical or agricultural products, or foodsor beverages, and ordinary acids and bases. Previously employedindustrial processes for production of nematocidal preparations arechemical syntheses which use dangerous and toxic starting materials andresult in waste streams of high toxicity which must be disposed of.

The method involves a post-treatment of a fermentation material, whichcan be industrial fermentation waste, to produce a nematocidalcomposition.

As used herein the terms “nematocide” or “nematocidal”, and the phrases“prevent plant damage” and “control of growth”, with respect tonematodes, include not only the rapid, direct killing of nematodes, butalso the concept of repelling nematodes, the prevention or effectivecontrol of their multiplication or reproduction, the prevention ofnematode egg hatching, and confusing or immobilizing the nematodes sothat they are prevented from finding a mate or a plant to parasitize.

The methods of using the compositions of this invention for nematodecontrol are by application to any field, fruit, vegetable, floral orornamental crop or nursery crop that is sensitive to attack by plantparasitic nematodes, particularly the Meloidogyne species. Methods ofapplication are well-known in the art and include direct application tothe soil, either as a liquid or a dried solid, controlled release of thebioactive components from solid formulations into the surrounding soil,application to the plant roots directly before planting in the soil,foliar application and the like.

The term “soil”, used herein is intended to include all media capable ofsupporting the growth of plants and may include humus, sand, silt, loam,manure, compost and commercial potting mixtures among others.

The term “fermentor”, as used herein refers to apparatus used forvarious types of fermentation methods including, but not limited to,shaken culture, solid-state, continuous and batch fed methods that arecontemplated for production of the fermentation broths of this inventionin both laboratory and large scale fermentation processes.

The term “biologically acceptable acid”, as used herein refers to acidssuch as sulfuric acid, phosphoric acid, hydrochloric acid, acetic acidor formic acid.

The term “biologically acceptable alkali or base”, as used herein refersto bases such as sodium hydroxide, potassium hydroxide, magnesium oxide,magnesium hydroxide, calcium oxide, calcium hydroxide or ammoniumhydroxide.

The process of this invention may utilize various media for the initialculture growth and can consist of potato-dextrose agar, hay infusionagar, corn meal agar, leaf litter agar, PCNB agar, soil infusion(modified), or Yeast Malt Agar as are defined in the Manual ofIndustrial Microbiology and Biotechnology, Demain and Solomon, AmericanSociety of Microbiology, Washington, D.C., 1986.

According to one embodiment of this invention, fermentation is carriedout in shake-flasks or in stationary-vat fermentors. In shake-flasks,aeration is provided by agitation of the flask which causes mixing ofthe medium with air. In the stationary fermentors, agitation is providedby impeller means such as a disc turbine, vaned disc, open turbine, ormarine propeller; and aeration is accomplished by injecting air oroxygen into the fermentation mixture.

The fermentation medium consists of suitable sources of carbon,nitrogen, inorganic salts, and growth factors assimilable by themicroorganism. Suitable examples of carbon sources are various sugarssuch as dextrose, glucose, lactose, and maltose, starch, dextrin, cornmeal and glycerol.

The sources of nitrogen can be of organic, inorganic or mixedorganic/inorganic origin. Examples of nitrogen sources that can be usedin the culture medium are soybean meal, corn steep liquor, peanut meal,cottonseed meal, corn germ meal, fish meal, lard water, and variousammonium salts.

The inclusion of certain amounts of minerals and growth factors in thefermentation medium is also helpful. Crude medium ingredients such asdistillers' solubles, corn steep liquor, fish meal, yeast products,peptonized milk and whey contain not only minerals but growth factors.However, inorganic salts such as potassium phosphate, sodium chloride,ferric sulfate, calcium carbonate, cobalt chloride, magnesium sulfate,and zinc sulfate can be added to the fermentation medium.

Solid materials, such as calcium carbonate may be added in this process,to help with pH control, which sometimes favors particular types ofpellet formation for best results.

The process of producing the fermentation materials for use in thisinvention, while utilizing a shaken culture fermentation technique mayalso use such a technique for the initial stages or inoculum productionas well. Production cultures are started from specially grown inocula.Growth is generally rapid at first. It then slows down and a stationaryphase is usually reached. The production yield depends on the quantityof cells present, their specific activity, and the span of theirproduct-forming capacity.

The inoculum is placed in a liquid medium which is selected empiricallyfor its ability to allow the recovery of the majority of the cells inthe population. The spores produced on an initial growth medium, such aspotato-dextrose agar, are transferred into growth medium contained in aflask (tenned seed flask), that would allow for the germination andinitial growth of culture. The germinated spores in an active growthstate are then transferred to Erlenmeyer flasks (shake-flasks) orstationary-vat fermentors containing the specific fermentation medium. A1-2% inoculum is typically produced for the fermentation stage ofdevelopment.

The inoculum medium is within the purview of those skilled in the art,and additional information may be found in the Manual of IndustrialMicrobiology and Biotechnology, pages 31-40, supra.

A wide range of shaker-culture apparatus may be used in the practice ofthis invention. The main types of apparatus are based on either rotaryor reciprocating shaking machines. The process herein preferably usesrotary shakers in which the flasks move in orbits of about 50 mm atabout 200 to about 250 rpm, (but may vary between 100 and 500 rpm). Theculture moves smoothly around the inside of the flask (which is usuallyan Erlenmeyer flask). The scale-up of the fermentation process is wellknown to those skilled in the art.

The purpose of shaking in submerged culture is to supply oxygen andnutrients to the growing cells. In shaken cultures, the medium in thefermentation flasks is inoculated with cells or spores, as is the caseherein. The strain used as an inoculum is held as a master culture, inthe freeze-dried state or at reduced temperatures, such as −70° C. Theoptimal spore concentration to be used for the inoculum is easilydetermined by those skilled in the art by routine experimentation.

The biopesticidal compositions of the present invention can be usedagainst plant parasitic nematodes, including, for example, Meloidogynespp., Pratylenchus spp., Radopholus similis, Ditylenchus dipsaci,Heterodera spp., Xiphinema spp., Globodera spp. and Hoplolaemus spp.

The processed fermentation materials prepared according to the processof this invention can be used to control nematodes for a variety ofagricultural applications on many different plants and fruits including,but not limited to, artichokes, aubergines, banana, barley, beet roots,cacao, carrots, cassava, celery, chickpea, citrus, coconut, coffee, colecrops, corn, cotton, cowpea, eggplant, field bean, forages, ginseng,grape, guava, various lettuces, melons, millet, oat, okra, ornamentals,papaya, peanut, pepper, pigeon pea, pineapple, potatoes, rice, rye,sorghum, soybean, sugar beet, sugar cane, sweet peppers, sweet potato,tea, tobacco, tomatoes, turf, wheat and yam. Cultivated flowers can beprotected according to the present invention, such as carnations, rosebushes, gerberas, chrysanthemums, pot plants, philodendrons, ferns,figs, pothos, sanseverias, and cacti; examples of nursery plants wouldinclude all the ornamental and flowering shrubs.

The bioactive materials can be incorporated into the soil of flower potsor containers, by direct application to the area to be treated at thetime of planting, or up to several days earlier, or by application in acontrolled release form. Application to field or orchard crops can be bygranule dispersement on the surface with turnover of the soil by a clawcultivar or a light plow, generally to about 10 cm up to about 20 cmdepth of soil. As the nematocide is water soluble, a drip irrigationmethod for application is also possible.

The compositions of the present invention can be in a suitable form fordirect application or as a concentrate or primary composition whichrequires dilution with a suitable quantity of water or other diluentbefore application. The pesticidal concentration will vary dependingupon the nature of the particular formation, specifically whether it isa concentrate or to be used directly.

The nematocidally effective amount of the active materials will dependupon the population of the nematode expected to be encountered, thenematode type, soil, crop, and moisture. In general, the composition maybe applied at a field rate of from about 1 to about 200/lbs per acre;preferably at a rate of from about 5 to about 100/lbs per acre and mostpreferably at a rate of from about 10 to about 60 lbs/acre.

The nematocidal compositions may be in the form of a suspension, asolution, an emulsion, a dusting powder, a dispersible granule, awettable powder, an emulsifiable concentrate, an aerosol or impregnatedgranule, formulated by techniques well known to those skilled in theart.

Additives to these compositions may include surface active agents, inertcarriers, preservatives, humectants, feeding stimulants, attractants,encapsulating agents, binders, emulsifiers, dyes, U.V. protectants,buffers, flow agents, or other components which facilitate producthandling and application for protection against nematodes.

Examples of inert carriers include inorganic minerals such as kaolin,mica, gypsum, fertilizer, phyllosilicates, carbonates, sulfates, orphosphates; organic materials such as sugar, starches or cyclodextrins;or botanical materials such as wood products, cork, powdered corn cobs,rice hulls, peanut hulls and walnut shells.

Suitable surface active agents include anionic compounds such ascarboxylates, for example an alkali metal carboxylate of a long chainfatty acid; an N-acylsarcosinate; mono- or di-esters of phosphoric acidwith fatty alcohol ethoxylates or salts of such esters; fatty alcoholsulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate orsodium cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylatedalkylphenol sulfates; lignin sulfonates; petroleum sulfonates; alkylaryl sulfonates such as alkyl benzene sulfonates or lower alkylnaphthalene sulfonates such as butyl naphthalene sulfonate; salts orsulfonated naphthalene-formaldehyde condensates; salts of sulfonatedphenol-formaldehyde condensates or more complex sulfonates such as theamide sulfonates. Non-ionic agents include condensation products offatty acid esters, fatty alcohols, fatty acid amides or fatty alkyl- oralkenyl- substituted phenols with ethylene oxide, fatty esters ofpolyhydric alcohol ethers, such as sorbitan fatty acid esters,condensation products of such esters with ethylene oxide, such aspolyoxyethylene sorbitan fatty acid esters, block copolymers of ethyleneoxide and propylene oxide, and acetylenic glycols. Examples of cationicsurface active agents include an aliphatic mono-, di-, or polyamine asan acetate, naphthenate or oleate; an oxygen containing amine such as anamine oxide of polyoxyethylene alkylamine; an amide-linked amineprepared by the condensation of a carboxylic acid with a di- orpolyamine; or a quaternary ammonium salt.

It is also contemplated that the materials of this invention may be usedin combination with other essential biologicals or beneficialmicroorganisms or active ingredients, such as herbicides,anti-microbials, fungicides, insecticides, plant growth regulators ornutrients.

The compositions of this invention may also be formulated as activemixtures which may include finely divided dry or liquid diluents,extenders, fillers, conditioners, and excipients, including variousclays, diatomaceous earth, talc and the like, or water and variousorganic liquids and mixtures thereof.

Of course, the present invention is not limited to the particularembodiments and modes of operation described herein and it is possibleto imagine a number of variations in the details without departing fromthe scope of this invention.

The examples below are presented to describe preferred embodiments andutilities of the invention and are not meant to limit the inventionunless otherwise stated in the claims appended hereto.

EXAMPLE 1

Water-insoluble materials were isolated from a Myrothecium verrucariafermentation broth by centrifuging. These solids were washed byre-suspending in distilled water and centrifuged again. The washingprocess was repeated twice more, and the solids were recovered in powderform by freeze-drying. This powdered material was subsequently treatedby suspending a weighed amount of it in water and heating only, orheating within several acidic environments (as detailed in Table 1below). The materials resulting from these conditions were readjusted topH 4, freeze dried into a powder, and tested in a contact assay withRKN. All heat treatments were carried out at 121° C. for four hours inan autoclave. Groups of approximately 50 nematodes for each replicatewere incubated in 12½% aqueous suspensions of each of the reconstitutedmaterials for a period of 24 hours, then counted to determine thepercent mortality of RKN. The materials resulting from heating underacid conditions of pH 2 or lower showed significant increases innematocidal activity.

TABLE 1 Contact Nematocidal Assay for Fermentation Samples (InsolubleFermentation Fraction) Sample Treatment Average % Mortality of RKN¹ AHeated 5 B pH 3 + Heated 7 C pH 2 + Heated 88  D pH 1.5 + Heated 100  EpH 1.25 + Heated 100  F Untreated 5 ¹= Second stage juveniles of RKN,Meloidogyne incognita.

EXAMPLE 2

Filter cakes (washed, damp fermentation solids) from two fungalfermentations were re-suspended in three times their weight of water,adjusted to pH 2.0 with sulfuric acid and heated in an autoclave at 121°C. for three hours. The samples were cooled, re-adjusted to pH 4.05-4.10with sodium hydroxide solution and made up with distilled water to afinal weight eight times that of the cake used (12.5% concentration).Groups of approximately 50 root-knot nematodes for each replicate wereincubated for 24 hours in these suspensions as well as a distilled watercontrol and counted to determine mortality. The heat-treated fungalfermentation solids showed strong nematocidal activity, as indicated inTable 2.

TABLE 2 Nematodes Exposed to Average Percent Mortality sterile water 0heated Streptomyces erythraeus solids 97.2 heated Gibberella fujikuroisolids 98.5

EXAMPLE 3

Dry material was recovered by lyophilization from a suspension offermentation solids from an unclassified Streptomyces species. The solidmaterial (4.56 g) was re-suspended in 40 mL of water, adjusted to pH 1.9with sulfuric acid and heated at 121-122° C. for three hours. Aftercooling, the pH was re-adjusted to 4.0 with sodium hydroxide solution,giving a final suspension containing 9% by weight of the fermentationsolids. Groups of approximately 50 root-knot nematodes for eachreplicate were incubated for 24 hours with this suspension or with anantibiotic control solution (100 units of penicillin plus 0.1 mg ofstreptomycin per mL) and then counted to determine mortality. Theheat-treated fermentation solid suspension was effective in killing theplant-parasitic nematodes, as shown in Table 3.

TABLE 3 Average Percent Nematodes Exposed to Mortality Antibioticcontrol 0.4 Heated suspension of Streptomyces solids 100

EXAMPLE 4

Water-insoluble materials were isolated from a Myrothecium fermentationbroth by centrifuging as described in Example 1. This insoluble fractionwas acid treated by suspending a weighed amount in water and adjustingto pH 1.72. The sample was divided and one portion was heated at 120° C.for three hours while the other portion was kept at room temperature.The materials were both readjusted to pH 4 and lyophilized into apowder. The powders were reconstituted and used in a test measuringpercent mortality of root knot nematodes. Nematodes were incubated insolution for 24 hours, then counted to determine living and deadnematodes, as shown in Table 4. Reducing the pH to 1.72 and heatingincreased the percent mortality of root-knot nematodes.

TABLE 4 Effects of pH changes on contact nematocidal activity Avg. %Sample Treatment Mortality² Insoluble fermentation Heated at pH 1.7296.3 fraction Insoluble fermentation Unheated at pH 1.72 <1% fraction -Control ²= Activity against second-stage juveniles of RKN, Meloidogyneincognita

EXAMPLE 5 Effects of Acid Digestion on Nematocidal Activity ofFermentation Broths (Greenhouse Evaluation)

Whole myrothecium fermentation culture broth was divided into twoportions. One portion was treated by reducing the pH to 1.5 and heatingat 121° C. for two hours, then readjusting the pH to 4. The otherportion was an untreated control. The resulting materials werefreeze-dried into a powder. The powders were reconstituted in water toequal concentrations by weight and dosed at a rate of 1.0 gram/pot ontosmall soil pots containing cucumber seedlings and approximately 800root-knot nematodes per pot. The plants were maintained with light andwater for one week, then removed from the soil. The galls (rootswellings resulting from infection by root-knot nematode, Meloidogyneincognita) on each root were counted to determine the rate of nematodeinfection. Efficacy is measured by the percent gall reduction from theuntreated, nematode infected control plants. As shown in Table 5, acidtreated and heated fermentation broth was effective at reducing thenematode infection by about 80%, whereas fermentation broth that was notacid treated and heated controlled about 30% of the galling.

TABLE 5 Sample % Gall Control Fermentation Broth (FB) 29.46 Acid TreatedFB 80.62

The above materials were also tested in a contact assay where root-knotnematodes were incubated for 24 hours in reconstituted solutions madefrom the lyophilized powders. Material that was acid treated and heatedincreased the percent mortality of the nematodes, whereas the untreatedbroth and broth that was heated without the acid treatment showed lowmortality of the nematodes, as shown in Table 6.

TABLE 6 Average % Treatment of RKN Mortality Fermentation Broth -unmodified 3.5 Fermentation Broth - heated 3.4 Fermentation Broth -heated under 100 acidic conditions

EXAMPLE 6 Nematocidal Activity of Acid-or Alkali Digested FermentationSamples (Contact Assay)

Myrothecium fermentation broth was heated in acid and basic (alkaline)conditions, and the efficacy of the resulting materials was compared ina nematocidal assay (% mortality of root knot nematodes). The controlbroth sample was adjusted to neutral pH (pH 7), and other samples wereadjusted to acidic (pH 2) and basic (pH 12) conditions. Half of eachsample was heated at 121° C. for two hours. Once cooled, all the sampleswere readjusted to neutral pH and lyophilized (dried to a powder). Thepowders were reconstituted in water to equal concentrations by weightand tested in a contact assay. The contact assay consisted of RKNincubated for 24 hours in five concentrations of each material, withmultiple replications. The nematodes were counted, and the number ofdead nematodes determined for each concentration for every sample. Datafrom this test was analyzed by PROBIT analysis to determine the LC₅₀(lethal concentration or dose to obtain an estimated 50% mortality ofthe nematodes within 95% confidence range). Fermentation broth whenheated in either acid or basic conditions showed increased toxicity tonematodes, indicated by a lower LC₅₀ value for these materials, as shownin Table 7. Materials that were pH adjusted but not heated did not showa significant increase of toxicity to nematodes.

TABLE 7 LC₅₀ against M. incognita Sample Unheated Heated FermentationBroth (FB) 3.59 5.54 pH 7 FB acid condition (pH 2) 3.58 1.01 FB alkalinecondition 2.52 0.88 (pH 12)

When plant parasitic nematodes are placed in direct contact with thecompositions so produced, the nematodes are paralyzed or killed,depending on the concentration of the composition applied. When the soilin which potted plants are growing is treated with a suspension of theproduct produced according to the method of this invention, and theninoculated with plant parasitic nematodes, after a growing period ofseveral days, the plants are healthier, with larger and heavier aerialparts than untreated plants similarly inoculated with nematodes. Theroots of the treated plants also exhibited fewer root galls (symptoms ofnematode attack and invasion) than the untreated plants. In the field,the composition produced may be applied as a suspension in water or as adry granular material to the plants to be protected from parasiticnematodes.

EXAMPLE 7

Portions of whole fermentation beer from the submerged fermentation ofMyrothecium sp. were adjusted to pH's of 4, 8, 9, 10 or 11 (±0.1) withsulfuric acid or sodium hydroxide and heated in an autoclave for twohours at 121° C. Control samples were pH adjusted but not heated. Afterheating, the samples were cooled to room temperature, and all sampleswere adjusted to pH 4.1 (±0.1) using sulfuric acid. Then samples werediluted to a final concentration of 2% solids. Groups of Meloidogyneincognita (root-know nematodes, RKN) were incubated in the suspensionsas well as a dilute antibiotic control solution (1 unit/mL of penicillinand 0.1 mg/mL of streptomycin), and after 24 hours live and deadindividuals were counted. Beer samples heated at elevated pHdemonstrated increased nematocidal activity over non-heated samples, asshown in Table 8.

TABLE 8 Whole-Beer Alkaline Heat Treatment Percent Mortality of RKN at2% Concentration Heated Non-heated pH 4  8.3 5.7 pH 8  6.4 0.0 pH 9 18.51.8 pH 10 33.3 0.0 pH 11 32.3 2.1 antibiotic control NA 0.0 Allreferences cited are hereby incorporated by reference.

The present invention is illustrated by way of the foregoing descriptionand examples. The foregoing description is intended as a non-limitingillustration, since many variations will become apparent to thoseskilled in the art in view thereof. It is intended that all suchvariations within the scope and spirit of the appended claims beembraced thereby.

Changes can be made in the composition, operation and arrangement of themethod of the present invention described herein without departing fromthe concept and scope of the invention as defined in the followingclaims:

We claim:
 1. A composition comprising a heat-treated, pH adjustedfermentation broth of a fungus or bacterium selected from the groupconsisting of Gibberella fujikuroi, Streptomyces erythraeus, Bacillussphaericus, Bacillus thuringiensis and Fusarium moniliforme; whereinsaid broth is pH adjusted either to a pH of below about 2.5 with abiologically acceptable acid, or to a pH of above about 8 with abiologically acceptable alkali or base; and wherein said broth is heatedto a temperature of at least about 100° C. for at least about 15minutes, then cooled to ambient temperature.